Heme-substituted protein assembly bridged by synthetic porphyrin: achieving controlled configuration while maintaining rotational freedom

The use of biological host–guest interactions, specifically the binding of hemoprotein to heme, has attracted significant research interest in the design of artificial protein assemblies. However, because of the inherent flexibility of the propionic acid group of heme, it is difficult to control the positioning and orientation of the protein unit and to construct well-ordered structures. Herein, we report a heme-substituted protein dimer composed of the native hemoprotein HasA, which accommodates a tetraphenylporphyrin bearing an additional metal coordination site. The specific binding of the tetraphenylporphyrin with an additional metal coordination site that protrudes in a fixed direction confines the configuration of the dimer structure to a defined bent form. The small-angle X-ray scattering profile shows the dimer structure with a bent form and suggests dynamic rotational behavior while keeping its bent-core structure, resembling a bevel gear. This unique dimer structure demonstrates that the design of heme-substituted protein assemblies can be expanded to protein assemblies while maintaining the rotational freedom of the individual protein units.

out using H buffer, and specific binding proteins including HasA were eluted with H buffer containing 200 mM imidazole.The resulting protein solution was treated with thrombin to remove the His-tag on HasA and dialyzed against a phosphate-buffered saline (PBS; 140 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.3)   solution overnight.This sample was further purified by the same Ni-affinity column chromatography equilibrated with PBS solution.The purified HasA solution was denatured by acetone including 0.2 % (w/v) conc.HCl to remove heme.The precipitate of HasA was collected by centrifugation and dissolved in a solution of 7 M urea including 100 mM Tris-HCl (pH7.5).The resulting solution was dialyzed against a PBS solution.After overnight dialysis, the solution was concentrated using Amicon Ultra (Merck Millipore, 3 kDa cutoff) and purified by a gel filtration column (HiPrep 16/60 Sephacryl S-200 HR; GE Healthcare, Cytiva) equilibrated with a PBS solution to remove unfolded HasA.The concentration of apo HasA was determined by absorption at 280 nm (HasAp: ε 280 = 28.6 mM -1 cm -1 , 5 HasApf5: ε 280 = 28.5 mM -1 cm -1 estimated via a Pierce TM BCA Protein Assay Kit (Thermo Scientific) using HasAp as the protein standard).The purified apo HasA solution was frozen by liquid nitrogen and stored at -80 °C until use.

Preparation of HasApf5 with Fe-salophen
The salophen ligand and its iron complex (Fe-salophen Cl) were prepared according to a method described in the literature. 1,6  methanol solution of Fe-salophen•Cl was added to apo-HasApf5 in a PBS solution on ice and gently stirred at 4 °C.Following overnight dialysis of a mixture of Fe-salophen/HasApf5 in PBS, excess Fe-salophen was removed by filtration through a PVDF membrane (0.22 µm, Millipore).The resulting HasApf5 solution was loaded on an anion exchange column (HiTrap capto DEAE; GE Healthcare, Cytiva) equilibrated with buffer A [50 mM Tris-HCl (pH 7.5)] and washed by 1 column volume of the buffer A containing 10%(v/v) of buffer B [50 mM Tris-HCl, 800 mM NaCl (pH 7.5)].The binding proteins to the anion exchange column were then eluted with 16 column volumes of a linear gradient from 10% to 80% as the ratio of buffer B, and we collected the pure Fe-salophen coordinating HasApf5 solution.After buffer exchange to PBS solution using desalting column (PD-10; GE Healthcare, Cytiva), the HasApf5 with Fe-salophen solution was frozen by liquid nitrogen and stored at 4 °C or -80 °C until use.

Preparation of HasApf5 with Fe-Pc
Fe(II) phthalocyanine (Fe-Pc) was purchased from TCI and used without further purification.Fe-Pc was solubilized in DMSO and then mixed with apo HasApf5 in PBS.Following overnight dialysis of a mixture of Fe-Pc/HasApf5 in PBS, excess Fe-Pc was removed by filtration through a PVDF membrane (0.22 µm, Millipore).The resulting HasApf5 solution was loaded on an anion exchange column (HiTrap capto DEAE; GE Healthcare, Cytiva) equilibrated with buffer A [50 mM Tris-HCl (pH 7.5)] and washed by 1 column volume of the buffer A containing 10%(v/v) of buffer B [50 mM Tris-HCl, 800 mM NaCl (pH 7.5)].The binding proteins to the anion exchange column were then eluted with 16 column volumes of a linear gradient from 10% to 80% as the ratio of buffer B, and we collected the pure Fe-Pc coordinating HasApf5 solution.After buffer exchange to PBS solution using desalting column (PD-10; GE Healthcare, Cytiva), the HasApf5 with Fe-Pc solution was frozen by liquid nitrogen and stored at 4 °C or -80 °C until use.

Analysis of Fe-TPP HasApf5 stability via UV-Vis spectroscopy
After buffer exchange to 0.1 M KPi (pH 7.0) or 0.1 M CHES-KOH (pH 9.5) using desalting column (PD-10; GE Healthcare, Cytiva), the concentration of Fe-TPP HasApf5 solutions was adjusted to 15 μM.The solutions of Fe-TPP HasApf5 were incubated at 4 °C and UV-Vis spectra were measured every 24 h after centrifugation (11,000 rpm, 10 min).

Circular Dichroism (CD) Spectroscopy
The thermal stability of HasApf5 capturing Fe-TPP was analyzed by CD spectroscopy (J-1500; JASCO).Molar ellipticity at 222 nm was measured as a function of temperature from 35 °C to 95 °C at increments of 1.0 °C min −1 controlled by a Peltier thermostat.Temperatures upon thermal denaturation (T m ) were determined by a previously reported method. 8

Crystallization of HasApf5 capturing heme
Purified holo HasApf5 was exchanged into a crystallization buffer [0.1 M KPi buffer (pH 7.0)] via a PD-10 desalting column (GE Healthcare, Cytiva).The eluate was concentrated via an Amicon Ultra filter 3 kDa cutoff (Merck Millipore).The final concentration was determined by UV-Vis spectroscopy.The crystallization condition was obtained from screening tests using Wizard Classic 1 and 2 (Rigaku).Crystals were grown at 20 °C using a sittingdrop vapor-diffusion method mixing 1 µL of HasApf5 with an equal volume of reservoir solution.For the X-ray diffraction study, crystals were flash-cooled in liquid nitrogen after soaking in a solution containing 70-80% suitable reservoir solution and 20-30% glycerol.The final crystallization condition was as follows: Crystallization condition of holo HasApf5.

Data collection and structure refinement of holo HasApf5
X-ray diffraction data was recorded on the beamline BL26B1 equipped with EIGER X 4M (Dectris) at SPring-8 (Hyogo, Japan) with a wavelength of 1.000 Å at 100 K. Reflection data of holo-HasApf5 was integrated by the program XDS 9 and scaled with Aimless [10][11][12] .Initial phases were determined by molecular replacement with MolRep 13,14 .The SWISS-MODEL structure of HasApf5 with heme was modeled from the crystal structure of holo HasAp (PDB ID: 3ELL 15 ) and used as a search model for MolRep 14 .Model building and refinement were performed by using Coot 16 and REFMAC5 17 .All protein figures were depicted by using PyMOL2.The final refinement statistics are summarized in Table.S3.  (2.96 ml) and CH 3 CN (5.93 ml).After the bubbling of argon, the solution was refluxed for 1.5 h.CH 2 Cl 2 (10 ml) was added, and the organic phase was washed with 2 M aqueous HCl solution and water.The resulting solution was dried over Na 2 SO 4 and removed the solvent under reduced pressure.The residue was redissolved with CH 2 Cl 2 (4.5 ml) and added EDTA buffer (4.5 ml) prepared by mixing Na 2 (H 2 EDTA) 2 H 2 O (372 mg), sodium acetate (0.22 g) and acetic acid (0.21 mL) in water (100 mL). 20The solution was stirred for 24 hours at room temperature.The organic phase was separated, washed with 4% sodium bicarbonate solution, 2 M aqueous HCl solution, and water, dried over Na 2 SO 4, and removed the solvent under reduced pressure.Fe-TPP-phen was given as a dark brown solid (14.5 mg, 15.7 μmol, 77% yield).MALDI-TOF MS: m/z calced.for C57H35ClFeN7O [M−Cl] + 889.2, found 889.8.

Fig
Fig. S2 Time-dependent changes in UV-Vis spectra of Fe-TPP HasApf5 in alkaline and neutral buffer.The graph shows the changes in the ratio of Fe-TPP bound form to the remaining HasApf5.

Fig
Fig. S3 Positive-mode ESI-TOF mass spectra and UV-Vis spectra of HasApf5s with Fe-salophen, or Fe-Pc.The structures of the metal complexes and pictures of the protein solutions in plastic containers are shown as insets.

Fig
Fig. S5 SEC analysis of the Fe-TPP-phen HasApf5 with Ni 2+ and EDTA.Following incubation of the solution of Fe-TPP-phen HasApf5 with Ni 2+ (0.5 equiv.)for 24 hours at 4 °C, then 50 equiv.EDTA was added, and the mixture was analyzed after incubation for 24 hours.Fe-TPP-phen HasApf5 with Ni 2+ and EDTA (red), and only Ni 2+ (gray).

Fig
Fig. S8 SEC analysis of the solution of Fe-TPP-phen HasApf5 with Ni 2+ in 50 mM CHES-KOH pH 9.5.Following incubation of the solution of Fe-TPP-phen HasApf5 with Ni 2+ (0.5 equiv.) in 50 mM CHES-KOH pH 9.5 for 24 hours, the mixture was analyzed (gray).In addition, the fraction containing the dimer in 50 mM CHES-KOH pH 9.5 was collected and concentrated.Following incubation of the concentrated solution for 24 hours at 4 °C, the solution was analyzed again (red).

Fig
Fig. S9 SEC analysis of the solution of Fe-TPP-phen HasApf5 with Ni 2+ in 50 mM KPi pH 7.0.Following incubation of the solution of Fe-TPP-phen HasApf5 with Ni 2+ (0.5 equiv.) in 50 mM CHES-KOH pH 9.5 for 24 hours, the mixture was analyzed (blue).In addition, the fraction containing the dimer in 50 mM KPi pH 7.0 was collected and concentrated.Following incubation of the concentrated solution for 24 hours at 4 °C, the solution was analyzed again (red).Furthermore, the solution of Fe-TPP-phen HasApf5 was also analyzed (gray).

Fig
Fig. S10 (a) Distribution plots of I(0) and R g obtained from Guinier analysis of all SEC-SAXS data.R g values between 227th and 286th data used to derive the SAXS profile are plotted.(b) Acquired SAXS experimental profile of the resulting dimer.(c) Guinier plot for (b).The red line represents the result of the linear approximation in the Guinier analysis.The residuals of the linear approximation are shown at the bottom of the graph.(d) Dimensionless Kratky plot of (b).(e) P(r) function.

Fig. S14
Fig. S14 Energy diagram of Λ-trans1 with changing the dihedral angle between the amide bond and the phenyl group of Fe-TPP-phen.

Fig. S15
Fig. S15 Crystal structure of holo HasApf5 and enlarged view of the heme-binding site of HasApf5.Polder map omitting heme contoured at 5.0σ (blue mesh).

Fig. S16
Fig. S16 Comparison of amino acid sequences between HasAp and HasApf5

Table . S3
Summary of crystallographic parameters